Process for the production of amino acids with coryneform bacteria using phosphoglucose isomerases from coryneform bacteria

ABSTRACT

The invention relates to polynucleotide sequences of pgi genes encoding polypeptide sequences having phosphoglucose isomerase activity from coryneform bacteria, to coryneform bacteria containing such polynucleotides and/or polypeptides, a process for the production of L-amino acids using such polynucleotides and/or polypeptides, and methods of screening and amplifying polynucleotides encoding polypeptide sequences which comprise varying degrees of phosphoglucose isomerase activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. application Ser. No. 60/379,391, filed May 13, 2002. The entire content of this application is incorporated herein by reference.

BACKGROUND OF INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates to polynucleotide sequences of pgi genes encoding polypeptide sequences having phosphoglucose isomerase activity from coryneform bacteria, to coryneform bacteria containing such polynucleotides and/or polypeptides, a process for the production of L-amino acids using such polynucleotides and/or polypeptides, and methods of screening and amplifying polynucleotides encoding polypeptide sequences which comprise varying degrees of phosphoglucose isomerase activity.

[0004] 2. Discussion of the Background

[0005] L-amino acids, especially L-lysine, may be used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and in the feeding of animals.

[0006] Amino acids may be produced by fermentation of strains of coryneform bacteria, especially Corynebacterium glutamicum. Because of their great importance, attempts are continuously being made to improve the production processes. Improvements to the processes may include measures relating to the fermentation. Examples of such improvements include stirring and oxygen supply, or improvements in the composition of the nutrient media. Examples of such improvements to the nutrient media include the sugar concentration during the fermentation. Also, improvements may include improving the work up of the product form by ion-exchange chromatography. Finally, improvements may include those of the intrinsic performance properties of the microorganism itself.

[0007] In order to improve the performance properties of such microorganisms, methods of mutagenesis, selection and mutant selection may be employed. Such methods yield strains which may be resistant to antimetabolites. Examples of such antimetabolites include the lysine analogue S-(2-aminoethyl)-cysteine. Further, some methods yield auxotrophic strains for metabolites that are important in terms of regulation, and which produce L-amino acids.

[0008] For a number of years, methods of recombinant DNA technology have also been used for improving the strain of L-amino acid-producing strains of Corynebacterium glutamicum, by amplifying or attenuating individual amino acid biosynthesis genes and studying the effect on L-amino acid production.

[0009] The nucleotide sequence of the chromosome of Corynebacterium glutamicum belongs to the prior art and has been published, for example, within the scope of EP-A-1108790 and WO 01/00844.

[0010] Studies relating to the pgi gene and the enzyme phosphoglucose isomerase coded for by that gene are described in EP-A-1087015 and in WO 01/07626.

SUMMARY OF THE INVENTION

[0011] An object of the present invention is to provide novel measures for improved preparation of L-amino acids or amino acids where these amino acids include L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine, including their salts (such as monohydrochloride or lysine sulfate).

[0012] One object of the present invention is a novel process for improving fermentative preparation of the L-amino acids, L-Lysine in particular. This process includes enhanced bacteria, preferably from Coryneform bacteria, which express attenuated amounts of phosphoglucose isomerase activity, which is encoded by the pgi gene.

[0013] Another object of the present invention is to provide such a bacterium, preferably from Coryneform bacteria, which expresses attenuated and/or enhanced pgi gene products. Another e object of the present invention is to provide such a bacterium, preferably from Coryneform bacteria, which expresses attenuated phosphoglucose isomerase activity.

[0014] Another object of the present invention is to provide a polynucleotide sequence encoding a polypeptide sequence with phosphoglucose isomerase activity. One embodiment of such a sequence is the polynucleotide sequence of SEQ ID NO. 1.

[0015] Another object of the present invention is a method of making phosphoglucose isomerase activity or a polypeptide having phosphoglucose isomerase activity. One embodiment of such a sequence is the polypeptide sequence of SEQ ID NO. 2.

[0016] Another object of the present invention relates to polynucleotide sequences encoding polypeptides having phosphoglucose isomerase activity and having the N-terminus optionally being shortened by 1 or 2, from 3 to 17, from 18 to 21, from 22 to 36 or from 37 to 46 amino acid residues, and nucleotide sequences that code for the mentioned proteins.

[0017] Another object of the present invention relates to polynucleotide sequences encoding polypeptides having phosphoglucose isomerase activity and having the N-terminus optionally being shortened by 1 or 2, from 3 to 17, from 18 to 21, from 22 to 36 or from 37 to 46 amino acid residues and the C-terminus having an amino acid sequence selected from the group SEQ ID NO:2 corresponding to position 47 to 585, SEQ ID NO:4 corresponding to position 47 to 585 and SEQ ID NO:6 corresponding to position 47 to 585, and nucleotide sequences that code for the mentioned proteins.

[0018] Another object of the present invention relates to polynucleotide sequences encoding polypeptides having phosphoglucose isomerase activity and having an amino acid sequence selected from the group SEQ ID NO:2, SEQ ID NO:2 corresponding to position 2 to 585, SEQ ID NO:4, SEQ ID NO:4 corresponding to position 2 to 585, SEQ ID NO:6 and SEQ ID NO:6 corresponding to position 2 to 585, and nucleotide sequences from coryneform bacteria that code for the mentioned proteins shown in SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, including the variants arising from the degeneracy of the genetic code.

[0019] Other objects of the present invention include methods of detecting polynucleotides that are homologous to SEQ ID NO: 1 or those polynucleotides encoding polypeptides that have having phosphoglucose isomerase activity, methods of making such polynucleotides encoding such polypeptides, and methods of making such polypeptides.

[0020] The above descriptions highlight certain aspects and embodiments of the present invention. Additional objects, aspects, and embodiments of the present invention follow in the detailed description of the present invention considered together with the Figures.

DETAILED DESCRIPTION OF THE INVENTION

[0021] Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan of molecular biology.

[0022] “Isolated” refers to a material, i.e. a polynucleotide separated out of its natural environment.

[0023] “Polynucleotide” in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non-modified RNA or DNA or modified RNA or DNA. Polynucleotides, which comprise the sequences according to the invention, are furthermore suitable as primers, which code for phosphoglucose isomerase activity can be prepared by the polymerase chain reaction (PCR).

[0024] Such oligonucleotides which serve as probes or primers comprise at least 25, 26, 27, 28, 29 or 30, preferably at least 20, 21, 22, 23 or 24, very particularly preferably at least 15, 16, 17, 18 or 19 successive nucleotides. Oligonucleotides which have a length of at least 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or at least 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides are also suitable. Oligonucleotides with a length of at least 100, 150, 200, 250 or 300 nucleotides are optionally also suitable.

[0025] The expression “from coryneform bacteria” means that the corresponding proteins or nucleic acids come from coryneform bacteria or have their origin therein.

[0026] Nucleotide sequences that code for the proteins according to the invention having phosphoglucose isomerase activity can also be designated pgi genes or alleles.

[0027] The invention also provides bacteria, such as Corynebacterium glutamicum or Escherichia coli, which contain the nucleotide sequences, or pgi genes or alleles, according to the invention.

[0028] Likewise, vectors containing nucleotide sequences according to the invention are claimed.

[0029] The invention also provides coryneform bacteria in which proteins having phosphoglucose isomerase activity are present in enhanced or attenuated form, the mentioned proteins being characterised by a length of 585 amino acid residues, and the N-terminus of the mentioned proteins optionally being shortened by 1 or 2, from 3 to 17, from 18 to 21, from 22 to 36 or from 37 to 46 amino acid residues. The ranges for the number of amino acids shortened include all ranges and subranges, including 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, and 45 nucleotides.

[0030] The term “enhancement” or “enhance” in this connection describes the increase in the intracellular activity or concentration of one or more enzymes or proteins in a microorganism that are coded for by the corresponding DNA, by, for example, increasing the number of copies of the gene or genes, using a strong promoter or using a gene or allele that codes for a corresponding enzyme or protein having a high level of activity, and optionally by combining those measures.

[0031] By the measures of enhancement, especially overexpression, the activity or concentration of the corresponding protein is generally increased by all ranges and subranges, including 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500%, 1000%, and 2000%, based on that of the wild-type protein or the activity or concentration of the protein in the starting microorganism.

[0032] The term “attenuation” or “attenuate” in this context describes the diminution or exclusion of the intracellular activity or concentration of one or more enzymes or proteins in a microorganism that are coded for by the corresponding DNA, by, for example, using a weak promoter or using a gene or allele that codes for a corresponding enzyme having a low level of activity, or by inactivating the corresponding enzyme or protein or gene, and optionally by combining those measures.

[0033] By the measures of attenuation, the activity or concentration of the corresponding protein is generally lowered to from 0 to 75%, from 0 to 50%, from 0 to 25%, from 0 to 10% or from 0 to 5% of the activity or concentration of the wild-type protein, or of the activity or concentration of the protein in the starting microorganism. The ranges for the attenuation include all ranges and subranges, including 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5%.

[0034] Where L-amino acids or amino acids are mentioned hereinbelow, they are to be understood as being one or more amino acids. Examples of such amino acids, including their salts, may be selected from the group L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine.

[0035] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0036] Reference is made to standard textbooks of molecular biology that contain definitions and methods and means for carrying out basic scientific techniques, encompassed by the present invention. See, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989) and various references cited therein.

[0037] Finally, the invention also provides a process for the production of one or more L-amino acids, especially selected from the group L-lysine, L-valine, L-threonine and L-methionine, with coryneform bacteria, which process is characterised by the following steps:

[0038] a) enhancement of proteins having phosphoglucose isomerase activity in the coryneform bacteria,

[0039] b) fermentation of the bacteria obtained in step a),

[0040] c) concentration of the amino acids in the medium, or in the fermentation liquor, or in the cells of the bacteria, and

[0041] d) isolation of the amino acids, constituents of the fermentation liquor and/or the biomass in totality or in part (from ≧0 to 100%) optionally remaining in the product,

[0042] the mentioned proteins being characterised by a length of 585 amino acid residues, and the N-terminus of the mentioned proteins optionally being shortened by 1 or 2, from 3 to 17, from 18 to 21, from 22 to 36 or from 37 to 46 amino acid residues.

[0043] Enhancement of the mentioned proteins having phosphoglucose isomerase activity is preferably used when the amino acid to be produced has a high NADPH consumption in the scope of its biosynthesis. That is the case, for example, with the amino acids L-lysine, L-valine, L-threonine and L-methionine.

[0044] The invention further provides a process for the production of one or more L-amino acids, especially selected from the group L-tryptophan, L-phenylalanine and L-tyrosine, with coryneform bacteria, which process is characterised by the following steps:

[0045] a) attenuation of proteins having phosphoglucose isomerase activity in the coryneform bacteria,

[0046] b) fermentation of the bacteria obtained in step a),

[0047] c) concentration of the amino acids in the medium, or in the fermentation liquor, or in the cells of the bacteria, and

[0048] d) isolation of the amino acids, constituents of the fermentation liquor and/or the biomass in totality or in part (from ≧0 to 100%) optionally remaining in the product,

[0049] the mentioned proteins being characterised by a length of 585 amino acid residues, and the N-terminus of the mentioned proteins optionally being shortened by 1 or 2, from 3 to 17, from 18 to 21, from 22 to 36 or from 37 to 46 amino acid residues.

[0050] Attenuation of the mentioned proteins having phosphoglucose isomerase activity is preferably used when a metabolite of the pentose phosphate cycle is a precursor of the amino acid to be produced. Erythrose-4-phosphate, for example, is a metabolite of the pentose phosphate cycle and a precursor of the aromatic amino acids L-tryptophan, L-phenylalanine and L-tyrosine. Summaries of the metabolic reactions and metabolites of the pentose phosphate cycle are to be found in textbooks of microbiology and biochemistry, such as, for example, the textbook of G. Gottschalk “Bacterial Metabolism” (2nd ed., Springer-Verlag, New York, USA, 1986).

[0051] The coryneform bacteria used may already produce L-amino acids before the enhancement or attenuation of the proteins according to the invention having phosphoglucose isomerase activity.

[0052] The microorganisms provided by the present invention may be able to produce L-amino acids from glucose, saccharose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol or ethanol or from acetic acid or lactic acid. They may be representatives of coryneform bacteria especially of the genus Corynebacterium. In the case of the genus Corynebacterium, special mention may be made of the species Corynebacterium glutamicum, which is known to those skilled in the art for its ability to produce L-amino acids.

[0053] Examples of suitable strains of the genus Corynebacterium, especially of the species Corynebacterium glutamicum, are especially the known wild-type strains

[0054]Corynebacterium glutamicum ATCC13032,

[0055]Corynebacterium acetoglutamicum ATCC 15806,

[0056]Corynebacterium acetoacidophilum ATCC 13870,

[0057]Corynebacterium melassecola ATCC 17965,

[0058]Corynebacterium thermoaminogenes FERM BP-1539,

[0059]Corynebacterium efficiens DSM44549

[0060]Brevibacterium flavum ATCC14067,

[0061]Brevibacterium lactofermentum ATCC13869 and

[0062]Brevibacterium divaricatum ATCC14020

[0063] and L-amino acid-producing mutants or strains prepared therefrom. Examples of such mutants or strains prepared therefrom are the L-lysine-producing strains

[0064]Corynebacterium glutamicum FERM-P 1709,

[0065]Brevibacterium flavum FERM-P 1708,

[0066]Brevibacterium lactofermentum FERM-P 1712,

[0067]Corynebacterium glutamicum FERM-P 6463,

[0068]Corynebacterium glutamicum FERM-P 6464,

[0069]Corynebacterium glutamicum DSM 5715,

[0070]Corynebacterium glutamicum DM58-1 and

[0071]Corynebacterium glutamicum DSM12866

[0072] and/or the the L-tryptophan-producing strains

[0073]Corynebacterium glutamicum ATCC21850 and

[0074]Corynebacterium glutamicum KY9218(pKW9901).

[0075] Strains designated “ATCC” can be obtained from the American Type Culture Collection (Manassas, Va., USA). Strains designated “FERM” can be obtained from the National Institute of Advanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan). Strains designated “DSM” can be obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). The strain Corynebacterium glutamicum DM58-1 is described in EP-A-0358940. The strain Corynebacterium glutamicum KY9218 (pKW9901) is described in Ikeda et al. (Bioscience Biotechnology and Biochemistry 58 (4), 674-678 (1994)).

[0076] During work on the present invention, it was possible to identify proteins having phosphoglucose isomerase activity in Corynebacterium glutamicum, which are shown in SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6. N-terminal amino acids can be cleaved by enzymes proper to the host, so-called peptidases. A known enzyme is aminopeptidase, which cleaves N-terminal methionine.

[0077] For determining the N-terminal amino acid sequence of a protein such as phosphoglucose isomerase, the “His-tag” method can be used. In that method, the coding region of the corresponding gene is lengthened at the 3′ end generally by from 1 to 10, typically 6, histidine codons. To that end, the coding region is inserted into corresponding vectors, as are described in the prior art, for example in the Qiagen Product Guide 2002 from Qiagen GmbH (Hilden, Germany). It is also possible to prepare the corresponding gene by means of the polymerase chain reaction using oligonucleotide primers which contain the histidine codons, and then insert the gene lengthened by the histidine codons into a suitable vector. Expression of the protein preferably takes place in Escherichia coli or Corynebacterium glutamicum. The protein provided with histidine labelling is then purified from the crude extract by affinity chromatography and the N-terminus is determined by Edman degradation. Purification of the protein may also be carried out by two-dimensional gel chromatography.

[0078] It has also been found that an improvement in the lysine production of lysine-producing coryneform bacteria can be achieved when the proteins according to the invention having phosphoglucose isomerase activity are enhanced.

[0079] In order to achieve an enhancement, either the expression or the catalytic properties of the proteins according to the invention can be increased. The two measures are optionally combined.

[0080] In order to achieve an overexpression, the number of copies of the corresponding genes can be increased, or the promoter and regulation thregion or the ribosome binding site, which is located upstream of the structural gene, can be mutated. Expression cassettes inserted upstream of the structural gene have a similar effect. By means of inducible promoters it is additionally possible to increase the expression in the course of the production of amino acids by fermentation. Expression is also improved by measures to prolong the life of the m-RNA. Furthermore, the enzyme activity is also enhanced by preventing degradation of the enzyme protein. The genes or gene constructs may either be present in plasmids with different numbers of copies or be integrated and amplified in the chromosome. Alternatively, overexpression of the genes in question may also be achieved by changing the composition of the medium and the manner in which culturing is carried out.

[0081] One will find instructions thereon inter alia in Martin et al. (Bio/Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in European patent specification 0 472 869, in U.S. Pat. No. 4,601,893, in Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991), in Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in patent application WO 96/15246, in Malumbres et al. (Gene 134, 15-24 (1993)), in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), in Makrides (Microbiological Reviews 60:512-538 (1996)) and in known textbooks of genetics and molecular biology.

[0082] A common method of achieving an overexpression consists in the use of episomal plasmids. Suitable plasmids are those which are replicated in coryneform bacteria. Many known plasmid vectors, such as, for example, pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKEx1 (Eikmanns et al., Gene 102:93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107:69-74 (1991)), are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid vectors, such as, for example, those which are based on pCG4 (U.S. Pat. No. 4,489,160) or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)) or pAG1 (U.S. Pat. No. 5,158,891), may likewise be used.

[0083] Suitable vectors may be those plasmid vectors with the aid of which the process of gene amplification by integration into the chromosome can be applied, as has been described by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for the duplication or amplification of the hom-thrB operon. In the method according to Reinscheid et al., the complete gene is cloned into a plasmid vector that is able to replicate in a host (typically E. coli), but not in C. glutamicum. Suitable vectors are, for example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), pGEM-T (Promega Corporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269:32678-32684; U.S. Pat. No. 5,487,993), pCR®Blunt (Invitrogen, Groningen, Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpf et al., 1991, Journal of Bacteriology 173:4510-4516) or pBGS8 (Spratt et al., 1986, Gene 41: 337-342). The plasmid vector containing the gene to be amplified is then transferred to the desired strain of C. glutamicum by conjugation or transformation. The method of conjugation is described, for example, in Schäfer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods of transformation are described, for example, in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a cross-over occurrence, the resulting strain contains at least two copies of the gene in question.

[0084] In order to achieve an attenuation, either the expression or the catalytic properties of the proteins according to the invention can be reduced. The two measures are optionally combined.

[0085] Gene expression can be diminished by carrying out the culturing in a suitable manner or by genetic alteration (mutation) of the signal structures of gene expression. Signal structures of gene expression are, for example, repressor genes, activator genes, operators, promoters, attenuators, ribosome-binding sites, the start codon and terminators. The person skilled in the art will find information thereon, for example, in patent application WO 96/15246, in Boyd and Murphy (Journal of Bacteriology 170: 5949 (1988)), in Voskuil and Chambliss (Nucleic Acids Research 26: 3548 (1998), in Jensen and Hammer (Biotechnology and Bioengineering 58: 191 (1998)), in Pátek et al. (Microbiology 142: 1297 (1996)), and in known textbooks of genetics and molecular biology, such as, for example, the textbook of Knippers (“Molekulare Genetik”, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or that of Winnacker (“Gene und Klone”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990). Methods of “anti-sense” RNA technology can also be used.

[0086] Mutations that lead to a change in or diminution of the catalytic properties of enzyme proteins are known from the prior art; examples which may be mentioned are the works of Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Möckel (“Die Threonindehydratase aus Corynebacterium glutamicum: Aufhebung der allosterischen Regulation und Struktur des Enzyms”, Berichte des Forschungszentrums Jülichs, Jül-2906, ISSN09442952, Jülich, Germany, 1994). Summaries are to be found in known textbooks of genetics and molecular biology, such as, for example, that of Hagemann (“Allgemeine Genetik”, Gustav Fischer Verlag, Stuttgart, 1986).

[0087] Examples of mutations may be transitions, transversions, insertions and deletions of at least one base pair. Depending on the effect of the amino acid substitution on the enzyme activity, missense mutations or nonsense mutations may be included. As a result of nonsense mutations, sense codons are converted into stop codons and the translation breaks off prematurely. Insertions or deletions of at least one base pair in a gene lead to frame shift mutations, as a result of which incorrect amino acids are incorporated or the translation breaks off prematurely. Deletions of one or more codons typically lead to complete loss of enzyme activity.

[0088] The mentioned mutations are preferably incorporated into the nucleotide sequences of the genes which code for the N-terminus of the proteins according to the invention having phosphoglucose isomerase activity. The N-terminus includes especially the amino acid residues 1 to 22, 1 to 37 or 1 to 46, or 3 to 22, 3 to 37 or 3 to 46, or 18 to 22, 18 to 37 or 18 to 46, or 22 to 37, 22 to 46 or 37 to 46 of SEQ ID NO:2, 4 or 6. The ranges for the number of amino acids shortened include all ranges and subranges, including 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, and 45 nucleotides.

[0089] The polynucleotides according to the invention include a polynucleotide encoding a polypeptide having phosphoglucose isomerase activity and SEQ ID NO:2, 4 or 6 or a fragment prepared therefrom and also those which are at least 70% to 80%, preferably at least 81% to 85%, particularly preferably at least 86% to 90%, and very particularly preferably at least 91%, 93%, 95%, 97% or 99% identical to the polynucleotide according to SEQ ID NO. 1 or a fragment prepared therefrom. The ranges for the percent identical include all ranges and subranges, including 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, and 98%.

[0090] The present invention provides polynucleotides which comprise the sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, nucleic acids or polynucleotides or genes which code for phosphoglucose isomerase activity ase or to isolate those nucleic acids or polynucleotides or genes which have a high similarity with the sequence of the pgi gene. They are also suitable for incorporation into so-called “arrays”, “micro arrays” or “DNA chips” in order to detect and to determine the corresponding polynucleotides.

[0091] Polynucleotides, which comprise the sequences according to the invention, are furthermore suitable as primers, which code for phosphoglucose isomerase activity can be prepared by the polymerase chain reaction (PCR).

[0092] Such oligonucleotides which serve as probes or primers comprise at least 25, 26, 27, 28, 29 or 30, preferably at least 20, 21, 22, 23 or 24, very particularly preferably at least 15, 16, 17, 18 or 19 successive nucleotides. Oligonucleotides which have a length of at least 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or at least 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides are also suitable. Oligonucleotides with a length of at least 100, 150, 200, 250 or 300 nucleotides are optionally also suitable.

[0093] The polypeptides according to the invention include a polypeptide according to SEQ ID NO:2, 4 or 6 or a fragment prepared therefrom, in particular those with the biological activity of phosphoglucose isomerase, and also those which are at least 70% to 80%, preferably at least 81% to 85%, particularly preferably at least 86% to 90%, and very particularly preferably at least 91%, 93%, 95%, 97% or 99% identical to the polypeptide according to SEQ ID NO. 2 and have the activity mentioned. The ranges for the percent identical include all ranges and subranges, including 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, and 98%.

[0094] Coding DNA sequences, which result from SEQ ID NO. 1 by the degeneracy of the genetic code, are also a constituent of the invention. In the same way, DNA sequences, which hybridize with SEQ ID NO. 1 or parts of SEQ ID NO. 1, are a constituent of the invention. Conservative amino acid exchanges, such as e.g. exchange of glycine for alanine or of aspartic acid for glutamic acid in proteins, are furthermore known among experts as “sense mutations” which do not lead to a fundamental change in the activity of the protein, i.e. are of neutral function. It is furthermore known that changes on the N and/or C terminus of a protein cannot substantially impair or can even stabilize the function thereof. Information in this context can be found by the expert, inter alia, in Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), in O'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) and in known textbooks of genetics and molecular biology. Amino acid sequences, which result in a corresponding manner from SEQ ID NO. 2, are also a constituent of the invention.

[0095] In the same way, DNA sequences, which hybridize with SEQ ID NO. 1 or parts of SEQ ID NO. 1, are a constituent of the invention. Finally, DNA sequences, which are prepared by the polymerase chain reaction (PCR) using primers, which result from SEQ ID NO. 1, are a constituent of the invention. Such oligonucleotides typically have a length of at least 15 nucleotides.

[0096] The skilled artisan will find instructions for identifying DNA sequences by means of hybridization can be found by the expert, inter alia, in the handbook “The DIG System Users Guide for Filter Hybridization” from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology 41: 255-260 (1991)). The hybridization takes place under stringent conditions, that is to say only hybrids in which the probe and target sequence, i.e. the polynucleotides treated with the probe, are at least 70% identical are formed. It is known that the stringency of the hybridization, including the washing steps, is influenced or determined by varying the buffer composition, the temperature and the salt concentration. The hybridization reaction is preferably carried out under a relatively low stringency compared with the washing steps (Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996).

[0097] A 5×SSC buffer at a temperature of approx. 50° C.-68° C., for example, can be employed for the hybridization reaction. Probes can also hybridize here with polynucleotides, which are less than 70% identical to the sequence of the probe. Such hybrids are less stable and are removed by washing under stringent conditions. This can be achieved, for example, by lowering the salt concentration to 2×SSC and optionally subsequently 0.5×SSC (The DIG System User's Guide for Filter Hybridisation, Boehringer Mannheim, Mannheim, Germany, 1995) a temperature of approx. 50° C.-68° C. being established. It is optionally possible to lower the salt concentration to 0.1×SSC. Polynucleotide fragments which are, for example, at least 70% or at least 80% or at least 90% to 95% identical to the sequence of the probe employed can be isolated by increasing the hybridization temperature stepwise from 50° C. to 68° C. in steps of approx. 1-2° C. Further instructions on hybridization are obtainable on the market in the form of so-called kits (e.g. DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalogue No. 1603558).

[0098] A skilled artisan will find instructions for amplification of DNA sequences with the aid of the polymerase chain reaction (PCR) can be found by the expert, inter alia, in the handbook by Gait: Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).

[0099] Instructions for the production of such mutations are part of the prior art and can be found in known textbooks of genetics and molecular biology, such as, for example, the textbook of Knippers (“Molekulare Genetik”, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that of Winnacker (“Gene und Klone”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or that of Hagemann (“Allgemeine Genetik”, Gustav Fischer Verlag, Stuttgart, 1986).

[0100] The invention also provides nucleotide sequences that are substantially identical with the described nucleotide sequences. They include those nucleotide sequences which contain at least one further, especially conservative, amino acid substitution.

[0101] In the case of aromatic amino acids, the expression conservative substitution is used when phenylalanine, tryptophan and tyrosine are substituted for one another. In the case of hydrophobic amino acids, the expression conservative substitution is used when leucine, isoleucine and valine are substituted for one another. In the case of polar amino acids, the expression conservative substitution is used when glutamine and asparagine are substituted for one another. In the case of basic amino acids, the expression conservative substitution is used when arginine, lysine and histidine are substituted for one another. In the case of acid amino acids, the expression conservative substitution is used when aspartic acid and glutamic acid are substituted for one another. In the case of amino acids containing hydroxyl groups, the expression conservative substitution is used when serine and threonine are substituted for one another.

[0102] A common method of mutating genes of C. glutamicum is the method of gene disruption and gene replacement described by Schwarzer and Pühler (Bio/Technology 9, 84-87 (1991)).

[0103] In the method of gene disruption, a central portion of the coding region of the gene in question is cloned into a plasmid vector which is able to replicate in a host (typically E. coli), but not in C. glutamicum. Suitable vectors are, for example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), pK18mobsacB or pK19mobsacB (Jäger et al., Journal of Bacteriology 174: 5462-5465 (1992)), pGEM-T (Promega Corporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269:32678-32684; U.S. Pat. No. 5,487,993), pCR®Blunt (Invitrogen, Groningen, Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) or pEM1 (Schrumpf et al., 1991, Journal of Bacteriology 173:4510-4516). The plasmid vector containing the central portion of the coding region of the gene is then transferred to the desired strain of C. glutamicum by conjugation or transformation. The method of conjugation is described, for example, in Schäfer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods of transformation are described, for example, in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a cross-over occurrence, the coding region of the gene in question is disrupted by the vector sequence, and two incomplete alleles lacking the 3′- and the 5′-end, respectively, are obtained. That method has been used, for example, by Fitzpatrick et al. (Applied Microbiology and Biotechnology 42, 575-580 (1994)) to exclude the recA gene of C. glutamicum.

[0104] In the gene replacement method, a mutation, such as, for example, a deletion, insertion or base substitution, is produced in vitro in the gene in question. The allele that is produced is in turn cloned into a vector that is not replicative for C. glutamicum, and the latter is then transferred to the desired host of C. glutamicum by transformation or conjugation. After homologous recombination by means of a first cross-over occurrence effecting integration and by means of a suitable second cross-over occurrence effecting an excision in the target gene or in the target sequence, incorporation of the mutation or of the allele is achieved. That method has been used, for example, by Peters-Wendisch et al. (Microbiology 144, 915-927 (1998)) to exclude the pyc gene of C. glutamicum by means of a deletion.

[0105] In addition, it may be advantageous for the production of L-amino acids, in addition to the enhancement or attenuation of the proteins according to the invention having phosphoglucose isomerase activity or of the genes or nucleotide sequences coding therefor, to enhance, especially overexpress, one or more enzymes of the biosynthesis pathway in question, of glycolysis, of the anaplerotic pathway, of the citric acid cycle, of the pentose phosphate cycle, of amino acid export, and optionally regulatory proteins.

[0106] The use of endogenous genes or endogenous nucleotide sequences is preferred. “Endogenous genes” or “endogenous nucleotide sequences” are to be understood as meaning the genes or alleles or the nucleotide sequences present in the population of a species.

[0107] Accordingly, for the production of L-lysine, it is possible, in addition to enhancing the proteins according to the invention having phosphoglucose isomerase activity, to enhance, especially overexpress, one or more genes selected from the group

[0108] the gene lysC coding for a feed-back resistant aspartate kinase (Accession No.P26512, EP-B-0387527; EP-A-0699759; WO 00/63388),

[0109] the gene dapA coding for dihydrodipicolinate synthase (EP-B 0 197 335),

[0110] the gene lysE coding for the lysine export protein (DE-A-195 48 222),

[0111] the gene pyc coding for pyruvate carboxylase (WO 99/18228, U.S. Pat. No. 6,171,833),

[0112] the gene gdh coding for glutamate dehydrogenase (U.S. Pat. No. 6,355,454),

[0113] the gene zwf coding for glucose 6-phosphate dehydrogenase (JP-A-09224661),

[0114] the gene mqo coding for malate:quinone oxidoreductase (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)), and

[0115] the gene zwa1 coding for the Zwa1 protein (EP-A-1111062).

[0116] It can also be advantageous for the production of L-lysine, in addition to enhancing the proteins according to the invention having phosphoglucose isomerase activity, to attenuate, especially reduce or exclude the expression of, one or more genes selected from the group

[0117] the gene ccpA1 coding for catabolite control protein A (WO 02/18419),

[0118] the gene pck coding for phosphoenol pyruvate carboxykinase (EP-A-1094111),

[0119] the gene fda coding for fructose bisphosphate aldolase (Molecular Microbiology 3 (11), 1625-1637 (1989); ACCESSION Number X17313),

[0120] the gene zwa2 coding for the Zwa2 protein (EP-A-1106693).

[0121] The microorganisms produced according to the invention also form part of the invention and can be cultivated continuously or discontinuously by the batch process or by the fed batch or repeated fed batch process for the purposes of the production of L-amino acids. A summary of known cultivation methods is described in the textbook of Chmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook of Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

[0122] The culture medium to be used must meet the requirements of the strains in question in a suitable manner. Descriptions of culture media for various microorganisms are to be found in the handbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981).

[0123] There may be used as the carbon source sugars and carbohydrates, such as, for example, glucose, saccharose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as, for example, soybean oil, sunflower oil, groundnut oil and coconut oil, fatty acids, such as, for example, palmitic acid, stearic acid and linoleic acid, alcohols, such as, for example, glycerol and ethanol, and organic acids, such as, for example, acetic acid or lactic acid. Those substances may be used individually or in the form of a mixture.

[0124] There may be used as the nitrogen source organic nitrogen-containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour and urea, or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or in the form of a mixture.

[0125] There may be used as the phosphorus source phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium must also contain salts of metals, such as, for example, magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances, such as amino acids and vitamins, may be used in addition to the above-mentioned substances. Suitable precursors may also be added to the culture medium. The mentioned substances may be added to the culture in the form of a single batch, or they may be fed in in a suitable manner during the cultivation.

[0126] In order to control the pH value of the culture, basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acid compounds, such as phosphoric acid or sulfuric acid, are expediently used. In order to control the development of foam, anti-foams, such as, for example, fatty acid polyglycol esters, may be used. In order to maintain the stability of plasmids, suitable substances having a selective action, such as, for example, antibiotics, may be added to the medium. In order to maintain aerobic conditions, oxygen or gas mixtures containing oxygen, such as, for example, air, are introduced into the culture. The temperature of the culture is normally from 20° C. to 45° C. and preferably from 25° C. to 40° C. The culture is continued until the maximum amount of the desired product has formed. That aim is normally achieved within a period of from 10 hours to 160 hours.

[0127] Methods of determining L-amino acids are known from the prior art. The analysis may be carried out as described in Spackman et al. (Analytical Chemistry, 30, (1958), 1190-1206) by anion-exchange chromatography with subsequent ninhydrin derivatisation, or it may be carried out by reversed phase HPLC, as described in Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).

[0128] Numerous modifications and variations on the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the accompanying claims, the invention may be practiced otherwise than as specifically described herein.

[0129] The present application claims priority to German Application No. DE 102 20 574.4, filed May 8, 2002. The entire content of this application is incorporated herein by reference.

1 6 1 1758 DNA Corynebacterium glutamicum CDS (1)..(1755) 1 atg gcc acg tcg aaa agc agc cca ata aac gca cct aaa ttt gtc gtg 48 Met Ala Thr Ser Lys Ser Ser Pro Ile Asn Ala Pro Lys Phe Val Val 1 5 10 15 ttt ccc act ttg aac act ctt cga tgc gct tgg cca caa aag caa gct 96 Phe Pro Thr Leu Asn Thr Leu Arg Cys Ala Trp Pro Gln Lys Gln Ala 20 25 30 aac ctg aag atg tta ttt aac gac aat aaa gga gtt ttc atg gcg gac 144 Asn Leu Lys Met Leu Phe Asn Asp Asn Lys Gly Val Phe Met Ala Asp 35 40 45 att tcg acc acc cag gtt tgg caa gac ctg acc gat cat tac tca aac 192 Ile Ser Thr Thr Gln Val Trp Gln Asp Leu Thr Asp His Tyr Ser Asn 50 55 60 ttc cag gca acc act ctg cgt gaa ctt ttc aag gaa gaa aac cgc gcc 240 Phe Gln Ala Thr Thr Leu Arg Glu Leu Phe Lys Glu Glu Asn Arg Ala 65 70 75 80 gag aag tac acc ttc tcc gcg gct ggc ctc cac gtc gac ctg tcg aag 288 Glu Lys Tyr Thr Phe Ser Ala Ala Gly Leu His Val Asp Leu Ser Lys 85 90 95 aat ctg ctt gac gac gcc acc ctc acc aag ctc ctt gca ctg acc gaa 336 Asn Leu Leu Asp Asp Ala Thr Leu Thr Lys Leu Leu Ala Leu Thr Glu 100 105 110 gaa tct ggc ctt cgc gaa cgc att gac gcg atg ttt gcc ggt gaa cac 384 Glu Ser Gly Leu Arg Glu Arg Ile Asp Ala Met Phe Ala Gly Glu His 115 120 125 ctc aac aac acc gaa gac cgc gct gtc ctc cac acc gcg ctg cgc ctt 432 Leu Asn Asn Thr Glu Asp Arg Ala Val Leu His Thr Ala Leu Arg Leu 130 135 140 cct gcc gaa gct gat ctg tca gta gat ggc caa gat gtt gct gct gat 480 Pro Ala Glu Ala Asp Leu Ser Val Asp Gly Gln Asp Val Ala Ala Asp 145 150 155 160 gtc cac gaa gtt ttg gga cgc atg cgt gac ttc gct act gcg ctg cgc 528 Val His Glu Val Leu Gly Arg Met Arg Asp Phe Ala Thr Ala Leu Arg 165 170 175 tca ggc aac tgg ttg gga cac acc ggc cac acg atc aag aag atc gtc 576 Ser Gly Asn Trp Leu Gly His Thr Gly His Thr Ile Lys Lys Ile Val 180 185 190 aac att ggt atc ggt ggc tct gac ctc gga cca gcc atg gct acg aag 624 Asn Ile Gly Ile Gly Gly Ser Asp Leu Gly Pro Ala Met Ala Thr Lys 195 200 205 gct ctg cgt gca tac gcg acc gct ggt atc tca gca gaa ttc gtc tcc 672 Ala Leu Arg Ala Tyr Ala Thr Ala Gly Ile Ser Ala Glu Phe Val Ser 210 215 220 aac gtc gac cca gca gac ctc gtt tct gtg ttg gaa gac ctc gat gca 720 Asn Val Asp Pro Ala Asp Leu Val Ser Val Leu Glu Asp Leu Asp Ala 225 230 235 240 gaa tcc aca ttg ttc gtg atc gct tcg aaa act ttc acc acc cag gag 768 Glu Ser Thr Leu Phe Val Ile Ala Ser Lys Thr Phe Thr Thr Gln Glu 245 250 255 acg ctg tcc aac gct cgt gca gct cgt gct tgg ctg gta gag aag ctc 816 Thr Leu Ser Asn Ala Arg Ala Ala Arg Ala Trp Leu Val Glu Lys Leu 260 265 270 ggt gaa gag gct gtc gcg aag cac ttc gtc gca gtg tcc acc aat gct 864 Gly Glu Glu Ala Val Ala Lys His Phe Val Ala Val Ser Thr Asn Ala 275 280 285 gaa aag gtc gca gag ttc ggt atc gac acg gac aac atg ttc ggc ttc 912 Glu Lys Val Ala Glu Phe Gly Ile Asp Thr Asp Asn Met Phe Gly Phe 290 295 300 tgg gac tgg gtc gga ggt cgt tac tcc gtg gac tcc gca gtt ggt ctt 960 Trp Asp Trp Val Gly Gly Arg Tyr Ser Val Asp Ser Ala Val Gly Leu 305 310 315 320 tcc ctc atg gca gtg atc ggc cct cgc gac ttc atg cgt ttc ctc ggt 1008 Ser Leu Met Ala Val Ile Gly Pro Arg Asp Phe Met Arg Phe Leu Gly 325 330 335 gga ttc cac gcg atg gat gaa cac ttc cgc acc acc aag ttc gaa gag 1056 Gly Phe His Ala Met Asp Glu His Phe Arg Thr Thr Lys Phe Glu Glu 340 345 350 aac gtt cca atc ttg atg gct ctg ctc ggt gtc tgg tac tcc gat ttc 1104 Asn Val Pro Ile Leu Met Ala Leu Leu Gly Val Trp Tyr Ser Asp Phe 355 360 365 tat ggt gca gaa acc cac gct gtc cta cct tat tcc gag gat ctc agc 1152 Tyr Gly Ala Glu Thr His Ala Val Leu Pro Tyr Ser Glu Asp Leu Ser 370 375 380 cgt ttt gct gct tac ctc cag cag ctg acc atg gaa tca aat ggc aag 1200 Arg Phe Ala Ala Tyr Leu Gln Gln Leu Thr Met Glu Ser Asn Gly Lys 385 390 395 400 tca gtc cac cgc gac ggc tcc cct gtt tcc act ggc act ggc gaa att 1248 Ser Val His Arg Asp Gly Ser Pro Val Ser Thr Gly Thr Gly Glu Ile 405 410 415 tac tgg ggt gag cct ggc aca aat ggc cag cac gct ttc ttc cag ctg 1296 Tyr Trp Gly Glu Pro Gly Thr Asn Gly Gln His Ala Phe Phe Gln Leu 420 425 430 atc cac cag ggc act cgc ctt gtt cca gct gat ttc att ggt ttc gct 1344 Ile His Gln Gly Thr Arg Leu Val Pro Ala Asp Phe Ile Gly Phe Ala 435 440 445 cgt cca aag cag gat ctt cct gcc ggt gag cgc acc atg cat gac ctt 1392 Arg Pro Lys Gln Asp Leu Pro Ala Gly Glu Arg Thr Met His Asp Leu 450 455 460 ttg atg agc aac ttc ttc gca cag acc aag gtt ttg gct ttc ggt aag 1440 Leu Met Ser Asn Phe Phe Ala Gln Thr Lys Val Leu Ala Phe Gly Lys 465 470 475 480 aac gct gaa gag atc gct gcg gaa ggt gtc gca cct gag ctg gtc aac 1488 Asn Ala Glu Glu Ile Ala Ala Glu Gly Val Ala Pro Glu Leu Val Asn 485 490 495 cac aag gtc atg cca ggt aat cgc cca acc acc acc att ttg gcg gag 1536 His Lys Val Met Pro Gly Asn Arg Pro Thr Thr Thr Ile Leu Ala Glu 500 505 510 gaa ctt acc cct tct att ctc ggt gcg ttg atc gct ttg tac gaa cac 1584 Glu Leu Thr Pro Ser Ile Leu Gly Ala Leu Ile Ala Leu Tyr Glu His 515 520 525 atc gtg atg gtt cag ggc gtg att tgg gac atc aac tcc ttc gac caa 1632 Ile Val Met Val Gln Gly Val Ile Trp Asp Ile Asn Ser Phe Asp Gln 530 535 540 tgg ggt gtt gaa ctg ggc aaa cag cag gca aat gac ctc gct ccg gct 1680 Trp Gly Val Glu Leu Gly Lys Gln Gln Ala Asn Asp Leu Ala Pro Ala 545 550 555 560 gtc tct ggt gaa gag gat gtt gac tcg gga gat tct tcc act gat tca 1728 Val Ser Gly Glu Glu Asp Val Asp Ser Gly Asp Ser Ser Thr Asp Ser 565 570 575 ctg att aag tgg tac cgc gca aat agg tag 1758 Leu Ile Lys Trp Tyr Arg Ala Asn Arg 580 585 2 585 PRT Corynebacterium glutamicum 2 Met Ala Thr Ser Lys Ser Ser Pro Ile Asn Ala Pro Lys Phe Val Val 1 5 10 15 Phe Pro Thr Leu Asn Thr Leu Arg Cys Ala Trp Pro Gln Lys Gln Ala 20 25 30 Asn Leu Lys Met Leu Phe Asn Asp Asn Lys Gly Val Phe Met Ala Asp 35 40 45 Ile Ser Thr Thr Gln Val Trp Gln Asp Leu Thr Asp His Tyr Ser Asn 50 55 60 Phe Gln Ala Thr Thr Leu Arg Glu Leu Phe Lys Glu Glu Asn Arg Ala 65 70 75 80 Glu Lys Tyr Thr Phe Ser Ala Ala Gly Leu His Val Asp Leu Ser Lys 85 90 95 Asn Leu Leu Asp Asp Ala Thr Leu Thr Lys Leu Leu Ala Leu Thr Glu 100 105 110 Glu Ser Gly Leu Arg Glu Arg Ile Asp Ala Met Phe Ala Gly Glu His 115 120 125 Leu Asn Asn Thr Glu Asp Arg Ala Val Leu His Thr Ala Leu Arg Leu 130 135 140 Pro Ala Glu Ala Asp Leu Ser Val Asp Gly Gln Asp Val Ala Ala Asp 145 150 155 160 Val His Glu Val Leu Gly Arg Met Arg Asp Phe Ala Thr Ala Leu Arg 165 170 175 Ser Gly Asn Trp Leu Gly His Thr Gly His Thr Ile Lys Lys Ile Val 180 185 190 Asn Ile Gly Ile Gly Gly Ser Asp Leu Gly Pro Ala Met Ala Thr Lys 195 200 205 Ala Leu Arg Ala Tyr Ala Thr Ala Gly Ile Ser Ala Glu Phe Val Ser 210 215 220 Asn Val Asp Pro Ala Asp Leu Val Ser Val Leu Glu Asp Leu Asp Ala 225 230 235 240 Glu Ser Thr Leu Phe Val Ile Ala Ser Lys Thr Phe Thr Thr Gln Glu 245 250 255 Thr Leu Ser Asn Ala Arg Ala Ala Arg Ala Trp Leu Val Glu Lys Leu 260 265 270 Gly Glu Glu Ala Val Ala Lys His Phe Val Ala Val Ser Thr Asn Ala 275 280 285 Glu Lys Val Ala Glu Phe Gly Ile Asp Thr Asp Asn Met Phe Gly Phe 290 295 300 Trp Asp Trp Val Gly Gly Arg Tyr Ser Val Asp Ser Ala Val Gly Leu 305 310 315 320 Ser Leu Met Ala Val Ile Gly Pro Arg Asp Phe Met Arg Phe Leu Gly 325 330 335 Gly Phe His Ala Met Asp Glu His Phe Arg Thr Thr Lys Phe Glu Glu 340 345 350 Asn Val Pro Ile Leu Met Ala Leu Leu Gly Val Trp Tyr Ser Asp Phe 355 360 365 Tyr Gly Ala Glu Thr His Ala Val Leu Pro Tyr Ser Glu Asp Leu Ser 370 375 380 Arg Phe Ala Ala Tyr Leu Gln Gln Leu Thr Met Glu Ser Asn Gly Lys 385 390 395 400 Ser Val His Arg Asp Gly Ser Pro Val Ser Thr Gly Thr Gly Glu Ile 405 410 415 Tyr Trp Gly Glu Pro Gly Thr Asn Gly Gln His Ala Phe Phe Gln Leu 420 425 430 Ile His Gln Gly Thr Arg Leu Val Pro Ala Asp Phe Ile Gly Phe Ala 435 440 445 Arg Pro Lys Gln Asp Leu Pro Ala Gly Glu Arg Thr Met His Asp Leu 450 455 460 Leu Met Ser Asn Phe Phe Ala Gln Thr Lys Val Leu Ala Phe Gly Lys 465 470 475 480 Asn Ala Glu Glu Ile Ala Ala Glu Gly Val Ala Pro Glu Leu Val Asn 485 490 495 His Lys Val Met Pro Gly Asn Arg Pro Thr Thr Thr Ile Leu Ala Glu 500 505 510 Glu Leu Thr Pro Ser Ile Leu Gly Ala Leu Ile Ala Leu Tyr Glu His 515 520 525 Ile Val Met Val Gln Gly Val Ile Trp Asp Ile Asn Ser Phe Asp Gln 530 535 540 Trp Gly Val Glu Leu Gly Lys Gln Gln Ala Asn Asp Leu Ala Pro Ala 545 550 555 560 Val Ser Gly Glu Glu Asp Val Asp Ser Gly Asp Ser Ser Thr Asp Ser 565 570 575 Leu Ile Lys Trp Tyr Arg Ala Asn Arg 580 585 3 1758 DNA Corynebacterium glutamicum CDS (1)..(1755) 3 atg gcc acg tcg aaa agc agc cca ata aac gca cct aaa ttt gtc gtg 48 Met Ala Thr Ser Lys Ser Ser Pro Ile Asn Ala Pro Lys Phe Val Val 1 5 10 15 ttt ccc act ttg aac act ctt cga tgc gct tgg cca caa aag caa gct 96 Phe Pro Thr Leu Asn Thr Leu Arg Cys Ala Trp Pro Gln Lys Gln Ala 20 25 30 aac ctg aag atg tta ttt aac gac aat aaa gga gtt ttc atg gcg gac 144 Asn Leu Lys Met Leu Phe Asn Asp Asn Lys Gly Val Phe Met Ala Asp 35 40 45 att tcg acc acc cag gct tgg caa gac ctg acc gat cat tac tca aac 192 Ile Ser Thr Thr Gln Ala Trp Gln Asp Leu Thr Asp His Tyr Ser Asn 50 55 60 ttc cag gca acc act ctg cgt gaa ctt ttc aag gaa gaa aac cgc gcc 240 Phe Gln Ala Thr Thr Leu Arg Glu Leu Phe Lys Glu Glu Asn Arg Ala 65 70 75 80 gag aag tac acc ttc tcc gcg gct ggc ctc cac gtc gac ctg tcg aag 288 Glu Lys Tyr Thr Phe Ser Ala Ala Gly Leu His Val Asp Leu Ser Lys 85 90 95 aat ctg ctt gac gac gcc acc ctc acc aag ctc ctt gca ctg acc gaa 336 Asn Leu Leu Asp Asp Ala Thr Leu Thr Lys Leu Leu Ala Leu Thr Glu 100 105 110 gaa tct ggc ctt cgc gaa cgc att gac gcg atg ttt gcc ggt gaa cac 384 Glu Ser Gly Leu Arg Glu Arg Ile Asp Ala Met Phe Ala Gly Glu His 115 120 125 ctc aac aac acc gaa gac cgc gct gtc ctc cac acc gcg ctg cgc ctt 432 Leu Asn Asn Thr Glu Asp Arg Ala Val Leu His Thr Ala Leu Arg Leu 130 135 140 cct ccc gaa gct gat ctg tca gta gat ggc caa gat gtt gct gct gat 480 Pro Pro Glu Ala Asp Leu Ser Val Asp Gly Gln Asp Val Ala Ala Asp 145 150 155 160 gtc cac gaa gtt ttg gga cgc atg cgt gac ttc gct act gcg ctg cgc 528 Val His Glu Val Leu Gly Arg Met Arg Asp Phe Ala Thr Ala Leu Arg 165 170 175 tca ggc aac tgg ttg gga cac acc ggc cac acg atc aag aag atc gtc 576 Ser Gly Asn Trp Leu Gly His Thr Gly His Thr Ile Lys Lys Ile Val 180 185 190 aac att ggt atc ggt ggc tct gac ctc gga cca gcc atg gct acg aag 624 Asn Ile Gly Ile Gly Gly Ser Asp Leu Gly Pro Ala Met Ala Thr Lys 195 200 205 gct ctg cgt gca tac gcg acc gct ggt atc tca gca gaa ttc gtc tcc 672 Ala Leu Arg Ala Tyr Ala Thr Ala Gly Ile Ser Ala Glu Phe Val Ser 210 215 220 aac gtc gac cca gca gac ctc gtt tct gtg ttg gaa gac ctc gat gca 720 Asn Val Asp Pro Ala Asp Leu Val Ser Val Leu Glu Asp Leu Asp Ala 225 230 235 240 gaa tcc aca ttg ttc gtg atc gct tcg aaa act ttt acc acc cag gag 768 Glu Ser Thr Leu Phe Val Ile Ala Ser Lys Thr Phe Thr Thr Gln Glu 245 250 255 acg ctg tct aac gct cgt gca gct cgt gct tgg ctg gta gag aag ctc 816 Thr Leu Ser Asn Ala Arg Ala Ala Arg Ala Trp Leu Val Glu Lys Leu 260 265 270 ggt gaa gag gct gtc gcg aag cat ttc gtc gca gtg tcc acc aat gct 864 Gly Glu Glu Ala Val Ala Lys His Phe Val Ala Val Ser Thr Asn Ala 275 280 285 gaa aag gtc gca gag ttc ggt atc gac acg gac aac atg ttc ggc ttc 912 Glu Lys Val Ala Glu Phe Gly Ile Asp Thr Asp Asn Met Phe Gly Phe 290 295 300 tgg gac tgg gtc gga ggt cgt tac tcc gtg gac tcc gca gtt ggt ctt 960 Trp Asp Trp Val Gly Gly Arg Tyr Ser Val Asp Ser Ala Val Gly Leu 305 310 315 320 tcc ctc atg gca gtg atc ggc cct cgc gac ttc atg cgt ttc ctc ggt 1008 Ser Leu Met Ala Val Ile Gly Pro Arg Asp Phe Met Arg Phe Leu Gly 325 330 335 gga ttc cac gcg atg gat gaa cac ttc cgc acc acc aag ttc gaa gag 1056 Gly Phe His Ala Met Asp Glu His Phe Arg Thr Thr Lys Phe Glu Glu 340 345 350 aac gtt cca atc ttg atg gct ctg ctc ggt gtc tgg tac tcc gat ttc 1104 Asn Val Pro Ile Leu Met Ala Leu Leu Gly Val Trp Tyr Ser Asp Phe 355 360 365 tat ggt gca gaa acc cac gct gtc cta cct tat tcc gag gat ctc agc 1152 Tyr Gly Ala Glu Thr His Ala Val Leu Pro Tyr Ser Glu Asp Leu Ser 370 375 380 cgt ttt gct gct tac ctc cag cag ctg acc atg gaa tca aac ggc aag 1200 Arg Phe Ala Ala Tyr Leu Gln Gln Leu Thr Met Glu Ser Asn Gly Lys 385 390 395 400 tca gtc cac cgc gac ggc tcc cct gtt tcc act ggc act ggc gaa att 1248 Ser Val His Arg Asp Gly Ser Pro Val Ser Thr Gly Thr Gly Glu Ile 405 410 415 tac tgg ggt gag cct ggc aca aat ggc cag cac gct ttc ttc cag ctg 1296 Tyr Trp Gly Glu Pro Gly Thr Asn Gly Gln His Ala Phe Phe Gln Leu 420 425 430 atc cac cag ggc act cgc ctt gtt cca gct gat ttc att ggt ttc gct 1344 Ile His Gln Gly Thr Arg Leu Val Pro Ala Asp Phe Ile Gly Phe Ala 435 440 445 cgt cca aag cag gat ctt cct gcc ggt gag cgc acc atg cat gac ctt 1392 Arg Pro Lys Gln Asp Leu Pro Ala Gly Glu Arg Thr Met His Asp Leu 450 455 460 ttg atg agc aac ttc ttc gca cag acc aag gtt ttg gct ttc ggt aag 1440 Leu Met Ser Asn Phe Phe Ala Gln Thr Lys Val Leu Ala Phe Gly Lys 465 470 475 480 aac gct gaa gag atc gct gcg gaa ggt gtc gca cct gag ctg gtc aac 1488 Asn Ala Glu Glu Ile Ala Ala Glu Gly Val Ala Pro Glu Leu Val Asn 485 490 495 cac aag gtc atg cca ggt aat cgc cca acc acc acc att ttg gcg gag 1536 His Lys Val Met Pro Gly Asn Arg Pro Thr Thr Thr Ile Leu Ala Glu 500 505 510 gaa ctt acc cct tct att ctc ggt gcg ttg atc gct ttg tac gaa cac 1584 Glu Leu Thr Pro Ser Ile Leu Gly Ala Leu Ile Ala Leu Tyr Glu His 515 520 525 atc gtg atg gtt cag ggc gtg att tgg gac atc aac tcc ttc gac caa 1632 Ile Val Met Val Gln Gly Val Ile Trp Asp Ile Asn Ser Phe Asp Gln 530 535 540 tgg ggt gtt gaa ctg ggc aaa cag cag gca aat gac ctc gct ccg gct 1680 Trp Gly Val Glu Leu Gly Lys Gln Gln Ala Asn Asp Leu Ala Pro Ala 545 550 555 560 gtc tct ggt gaa gag gat gtt gac tcg gga gat tct tcc act gat tca 1728 Val Ser Gly Glu Glu Asp Val Asp Ser Gly Asp Ser Ser Thr Asp Ser 565 570 575 ctg att aag tgg tac cgc gca aat agg tag 1758 Leu Ile Lys Trp Tyr Arg Ala Asn Arg 580 585 4 585 PRT Corynebacterium glutamicum 4 Met Ala Thr Ser Lys Ser Ser Pro Ile Asn Ala Pro Lys Phe Val Val 1 5 10 15 Phe Pro Thr Leu Asn Thr Leu Arg Cys Ala Trp Pro Gln Lys Gln Ala 20 25 30 Asn Leu Lys Met Leu Phe Asn Asp Asn Lys Gly Val Phe Met Ala Asp 35 40 45 Ile Ser Thr Thr Gln Ala Trp Gln Asp Leu Thr Asp His Tyr Ser Asn 50 55 60 Phe Gln Ala Thr Thr Leu Arg Glu Leu Phe Lys Glu Glu Asn Arg Ala 65 70 75 80 Glu Lys Tyr Thr Phe Ser Ala Ala Gly Leu His Val Asp Leu Ser Lys 85 90 95 Asn Leu Leu Asp Asp Ala Thr Leu Thr Lys Leu Leu Ala Leu Thr Glu 100 105 110 Glu Ser Gly Leu Arg Glu Arg Ile Asp Ala Met Phe Ala Gly Glu His 115 120 125 Leu Asn Asn Thr Glu Asp Arg Ala Val Leu His Thr Ala Leu Arg Leu 130 135 140 Pro Pro Glu Ala Asp Leu Ser Val Asp Gly Gln Asp Val Ala Ala Asp 145 150 155 160 Val His Glu Val Leu Gly Arg Met Arg Asp Phe Ala Thr Ala Leu Arg 165 170 175 Ser Gly Asn Trp Leu Gly His Thr Gly His Thr Ile Lys Lys Ile Val 180 185 190 Asn Ile Gly Ile Gly Gly Ser Asp Leu Gly Pro Ala Met Ala Thr Lys 195 200 205 Ala Leu Arg Ala Tyr Ala Thr Ala Gly Ile Ser Ala Glu Phe Val Ser 210 215 220 Asn Val Asp Pro Ala Asp Leu Val Ser Val Leu Glu Asp Leu Asp Ala 225 230 235 240 Glu Ser Thr Leu Phe Val Ile Ala Ser Lys Thr Phe Thr Thr Gln Glu 245 250 255 Thr Leu Ser Asn Ala Arg Ala Ala Arg Ala Trp Leu Val Glu Lys Leu 260 265 270 Gly Glu Glu Ala Val Ala Lys His Phe Val Ala Val Ser Thr Asn Ala 275 280 285 Glu Lys Val Ala Glu Phe Gly Ile Asp Thr Asp Asn Met Phe Gly Phe 290 295 300 Trp Asp Trp Val Gly Gly Arg Tyr Ser Val Asp Ser Ala Val Gly Leu 305 310 315 320 Ser Leu Met Ala Val Ile Gly Pro Arg Asp Phe Met Arg Phe Leu Gly 325 330 335 Gly Phe His Ala Met Asp Glu His Phe Arg Thr Thr Lys Phe Glu Glu 340 345 350 Asn Val Pro Ile Leu Met Ala Leu Leu Gly Val Trp Tyr Ser Asp Phe 355 360 365 Tyr Gly Ala Glu Thr His Ala Val Leu Pro Tyr Ser Glu Asp Leu Ser 370 375 380 Arg Phe Ala Ala Tyr Leu Gln Gln Leu Thr Met Glu Ser Asn Gly Lys 385 390 395 400 Ser Val His Arg Asp Gly Ser Pro Val Ser Thr Gly Thr Gly Glu Ile 405 410 415 Tyr Trp Gly Glu Pro Gly Thr Asn Gly Gln His Ala Phe Phe Gln Leu 420 425 430 Ile His Gln Gly Thr Arg Leu Val Pro Ala Asp Phe Ile Gly Phe Ala 435 440 445 Arg Pro Lys Gln Asp Leu Pro Ala Gly Glu Arg Thr Met His Asp Leu 450 455 460 Leu Met Ser Asn Phe Phe Ala Gln Thr Lys Val Leu Ala Phe Gly Lys 465 470 475 480 Asn Ala Glu Glu Ile Ala Ala Glu Gly Val Ala Pro Glu Leu Val Asn 485 490 495 His Lys Val Met Pro Gly Asn Arg Pro Thr Thr Thr Ile Leu Ala Glu 500 505 510 Glu Leu Thr Pro Ser Ile Leu Gly Ala Leu Ile Ala Leu Tyr Glu His 515 520 525 Ile Val Met Val Gln Gly Val Ile Trp Asp Ile Asn Ser Phe Asp Gln 530 535 540 Trp Gly Val Glu Leu Gly Lys Gln Gln Ala Asn Asp Leu Ala Pro Ala 545 550 555 560 Val Ser Gly Glu Glu Asp Val Asp Ser Gly Asp Ser Ser Thr Asp Ser 565 570 575 Leu Ile Lys Trp Tyr Arg Ala Asn Arg 580 585 5 1758 DNA Corynebacterium glutamicum CDS (1)..(1755) 5 atg gcc acg tcg aaa agc agc cca ata aac gca cct aaa ttt gtc gtg 48 Met Ala Thr Ser Lys Ser Ser Pro Ile Asn Ala Pro Lys Phe Val Val 1 5 10 15 ttt ccc act ttg aac act ctt cga tgc gct tgg cca caa aag caa gct 96 Phe Pro Thr Leu Asn Thr Leu Arg Cys Ala Trp Pro Gln Lys Gln Ala 20 25 30 aac ctg aag atg tta ttt aac gac aat aaa gga gtt ttc atg gcg gac 144 Asn Leu Lys Met Leu Phe Asn Asp Asn Lys Gly Val Phe Met Ala Asp 35 40 45 att tcg acc acc cag gtt tgg caa gac ctg acc gat cat tac tca aac 192 Ile Ser Thr Thr Gln Val Trp Gln Asp Leu Thr Asp His Tyr Ser Asn 50 55 60 ttc cag gca acc act ctg cgt gaa ctt ttc aag gaa gaa aac cgc gcc 240 Phe Gln Ala Thr Thr Leu Arg Glu Leu Phe Lys Glu Glu Asn Arg Ala 65 70 75 80 gag aag tac acc ttc tcc gcg gct ggc ctc cac gtc gac ctg tcg aag 288 Glu Lys Tyr Thr Phe Ser Ala Ala Gly Leu His Val Asp Leu Ser Lys 85 90 95 aat ctg ctt gac gac gcc acc ctc acc aag ctc ctt gca ctg acc gaa 336 Asn Leu Leu Asp Asp Ala Thr Leu Thr Lys Leu Leu Ala Leu Thr Glu 100 105 110 gaa tct ggc ctt cgc gaa cgc att gac gcg atg ttt gcc ggt gaa cac 384 Glu Ser Gly Leu Arg Glu Arg Ile Asp Ala Met Phe Ala Gly Glu His 115 120 125 ctc aac aac acc gaa gac cgc gct gtc ctc cac acc gcg ctg cgc ctt 432 Leu Asn Asn Thr Glu Asp Arg Ala Val Leu His Thr Ala Leu Arg Leu 130 135 140 cct gcc gaa gct gat ctg tca gta gat ggc caa gat gtt gct gct gat 480 Pro Ala Glu Ala Asp Leu Ser Val Asp Gly Gln Asp Val Ala Ala Asp 145 150 155 160 gtc cac gaa gtt ttg gga cgc atg cgt gac ttc gct act gcg ctg cgc 528 Val His Glu Val Leu Gly Arg Met Arg Asp Phe Ala Thr Ala Leu Arg 165 170 175 tca ggc aac tgg ttg gga cac acc ggc cac acg atc aag aag atc gtc 576 Ser Gly Asn Trp Leu Gly His Thr Gly His Thr Ile Lys Lys Ile Val 180 185 190 aac att ggt atc ggt ggc tct gac ctc gga cca gcc atg gct acg aag 624 Asn Ile Gly Ile Gly Gly Ser Asp Leu Gly Pro Ala Met Ala Thr Lys 195 200 205 gct ctg cgt gca tac gcg acc gct ggt atc tca gca gaa ttc gtc tcc 672 Ala Leu Arg Ala Tyr Ala Thr Ala Gly Ile Ser Ala Glu Phe Val Ser 210 215 220 aac gtc gac cca gca gac ctc gtt tct gtg ttg gaa gac ctc gat gca 720 Asn Val Asp Pro Ala Asp Leu Val Ser Val Leu Glu Asp Leu Asp Ala 225 230 235 240 gaa tcc aca ttg ttc gtg atc gct tcg aaa act ttc acc acc cag gag 768 Glu Ser Thr Leu Phe Val Ile Ala Ser Lys Thr Phe Thr Thr Gln Glu 245 250 255 acg ctg tcc aac gct cgt gca gct cgt gct tgg ctg gta gag aag ctc 816 Thr Leu Ser Asn Ala Arg Ala Ala Arg Ala Trp Leu Val Glu Lys Leu 260 265 270 ggt gaa gag gct gtc gcg aag cac ttc gtc gca gtg tcc acc aat gct 864 Gly Glu Glu Ala Val Ala Lys His Phe Val Ala Val Ser Thr Asn Ala 275 280 285 gaa aag gtc gca gag ttc ggt atc gac acg gac aac atg ttc ggc ttc 912 Glu Lys Val Ala Glu Phe Gly Ile Asp Thr Asp Asn Met Phe Gly Phe 290 295 300 tgg gac tgg gtc gga ggt cgt tac tcc gtg gac tcc gca gtt ggt ctt 960 Trp Asp Trp Val Gly Gly Arg Tyr Ser Val Asp Ser Ala Val Gly Leu 305 310 315 320 tcc ctc atg gca gtg atc ggc cct cgc gac ttc atg cgt ttc ctc ggt 1008 Ser Leu Met Ala Val Ile Gly Pro Arg Asp Phe Met Arg Phe Leu Gly 325 330 335 gga ttc cac gcg atg gat gaa cac ttc cgc acc acc aag ttc gaa gag 1056 Gly Phe His Ala Met Asp Glu His Phe Arg Thr Thr Lys Phe Glu Glu 340 345 350 aac gtt cca atc ttg atg gct ctg ctc ggt gtc tgg tac tcc gat ttc 1104 Asn Val Pro Ile Leu Met Ala Leu Leu Gly Val Trp Tyr Ser Asp Phe 355 360 365 tat ggt gca gaa acc cac gct gtc cta cct tat tcc gag gat ctc agc 1152 Tyr Gly Ala Glu Thr His Ala Val Leu Pro Tyr Ser Glu Asp Leu Ser 370 375 380 cgt ttt gct gct tac ctc cag cag ctg acc atg gag acc aat ggc aag 1200 Arg Phe Ala Ala Tyr Leu Gln Gln Leu Thr Met Glu Thr Asn Gly Lys 385 390 395 400 tca gtc cac cgc gac ggc tcc cct gtt tcc act ggc act ggc gaa att 1248 Ser Val His Arg Asp Gly Ser Pro Val Ser Thr Gly Thr Gly Glu Ile 405 410 415 tac tgg ggt gag cct ggc aca aat ggc cag cac gct ttc ttc cag ctg 1296 Tyr Trp Gly Glu Pro Gly Thr Asn Gly Gln His Ala Phe Phe Gln Leu 420 425 430 atc cac cag ggc act cgc ctt gtt cca gct gat ttc att ggt ttc gct 1344 Ile His Gln Gly Thr Arg Leu Val Pro Ala Asp Phe Ile Gly Phe Ala 435 440 445 cgt cca aag cag gat ctt cct gcc ggt gag cgc acc atg cat gac ctt 1392 Arg Pro Lys Gln Asp Leu Pro Ala Gly Glu Arg Thr Met His Asp Leu 450 455 460 ttg atg agc aac ttc ttc gca cag acc aag gtt ttg gct ttc ggt aag 1440 Leu Met Ser Asn Phe Phe Ala Gln Thr Lys Val Leu Ala Phe Gly Lys 465 470 475 480 aac gct gaa gag atc gct gcg gaa ggt gtc gca cct gag ctg gtc aac 1488 Asn Ala Glu Glu Ile Ala Ala Glu Gly Val Ala Pro Glu Leu Val Asn 485 490 495 cac aag gtc gtg cca ggt aat cgc cca acc acc acc att ttg gcg gag 1536 His Lys Val Val Pro Gly Asn Arg Pro Thr Thr Thr Ile Leu Ala Glu 500 505 510 gaa ctt acc cct tct att ctc ggt gcg ttg atc gct ttg tac gaa cac 1584 Glu Leu Thr Pro Ser Ile Leu Gly Ala Leu Ile Ala Leu Tyr Glu His 515 520 525 acc gtg atg gtt cag ggc gtg att tgg gac atc aac tcc ttc gac caa 1632 Thr Val Met Val Gln Gly Val Ile Trp Asp Ile Asn Ser Phe Asp Gln 530 535 540 tgg ggt gtt gaa ctg ggc aaa cag cag gca aat gac ctc gct ccg gct 1680 Trp Gly Val Glu Leu Gly Lys Gln Gln Ala Asn Asp Leu Ala Pro Ala 545 550 555 560 gtc tct ggt gaa gag gat gtt gac tcg gga gat tct tcc act gat tca 1728 Val Ser Gly Glu Glu Asp Val Asp Ser Gly Asp Ser Ser Thr Asp Ser 565 570 575 ctg att aag tgg tac cgc gca aat agg tag 1758 Leu Ile Lys Trp Tyr Arg Ala Asn Arg 580 585 6 585 PRT Corynebacterium glutamicum 6 Met Ala Thr Ser Lys Ser Ser Pro Ile Asn Ala Pro Lys Phe Val Val 1 5 10 15 Phe Pro Thr Leu Asn Thr Leu Arg Cys Ala Trp Pro Gln Lys Gln Ala 20 25 30 Asn Leu Lys Met Leu Phe Asn Asp Asn Lys Gly Val Phe Met Ala Asp 35 40 45 Ile Ser Thr Thr Gln Val Trp Gln Asp Leu Thr Asp His Tyr Ser Asn 50 55 60 Phe Gln Ala Thr Thr Leu Arg Glu Leu Phe Lys Glu Glu Asn Arg Ala 65 70 75 80 Glu Lys Tyr Thr Phe Ser Ala Ala Gly Leu His Val Asp Leu Ser Lys 85 90 95 Asn Leu Leu Asp Asp Ala Thr Leu Thr Lys Leu Leu Ala Leu Thr Glu 100 105 110 Glu Ser Gly Leu Arg Glu Arg Ile Asp Ala Met Phe Ala Gly Glu His 115 120 125 Leu Asn Asn Thr Glu Asp Arg Ala Val Leu His Thr Ala Leu Arg Leu 130 135 140 Pro Ala Glu Ala Asp Leu Ser Val Asp Gly Gln Asp Val Ala Ala Asp 145 150 155 160 Val His Glu Val Leu Gly Arg Met Arg Asp Phe Ala Thr Ala Leu Arg 165 170 175 Ser Gly Asn Trp Leu Gly His Thr Gly His Thr Ile Lys Lys Ile Val 180 185 190 Asn Ile Gly Ile Gly Gly Ser Asp Leu Gly Pro Ala Met Ala Thr Lys 195 200 205 Ala Leu Arg Ala Tyr Ala Thr Ala Gly Ile Ser Ala Glu Phe Val Ser 210 215 220 Asn Val Asp Pro Ala Asp Leu Val Ser Val Leu Glu Asp Leu Asp Ala 225 230 235 240 Glu Ser Thr Leu Phe Val Ile Ala Ser Lys Thr Phe Thr Thr Gln Glu 245 250 255 Thr Leu Ser Asn Ala Arg Ala Ala Arg Ala Trp Leu Val Glu Lys Leu 260 265 270 Gly Glu Glu Ala Val Ala Lys His Phe Val Ala Val Ser Thr Asn Ala 275 280 285 Glu Lys Val Ala Glu Phe Gly Ile Asp Thr Asp Asn Met Phe Gly Phe 290 295 300 Trp Asp Trp Val Gly Gly Arg Tyr Ser Val Asp Ser Ala Val Gly Leu 305 310 315 320 Ser Leu Met Ala Val Ile Gly Pro Arg Asp Phe Met Arg Phe Leu Gly 325 330 335 Gly Phe His Ala Met Asp Glu His Phe Arg Thr Thr Lys Phe Glu Glu 340 345 350 Asn Val Pro Ile Leu Met Ala Leu Leu Gly Val Trp Tyr Ser Asp Phe 355 360 365 Tyr Gly Ala Glu Thr His Ala Val Leu Pro Tyr Ser Glu Asp Leu Ser 370 375 380 Arg Phe Ala Ala Tyr Leu Gln Gln Leu Thr Met Glu Thr Asn Gly Lys 385 390 395 400 Ser Val His Arg Asp Gly Ser Pro Val Ser Thr Gly Thr Gly Glu Ile 405 410 415 Tyr Trp Gly Glu Pro Gly Thr Asn Gly Gln His Ala Phe Phe Gln Leu 420 425 430 Ile His Gln Gly Thr Arg Leu Val Pro Ala Asp Phe Ile Gly Phe Ala 435 440 445 Arg Pro Lys Gln Asp Leu Pro Ala Gly Glu Arg Thr Met His Asp Leu 450 455 460 Leu Met Ser Asn Phe Phe Ala Gln Thr Lys Val Leu Ala Phe Gly Lys 465 470 475 480 Asn Ala Glu Glu Ile Ala Ala Glu Gly Val Ala Pro Glu Leu Val Asn 485 490 495 His Lys Val Val Pro Gly Asn Arg Pro Thr Thr Thr Ile Leu Ala Glu 500 505 510 Glu Leu Thr Pro Ser Ile Leu Gly Ala Leu Ile Ala Leu Tyr Glu His 515 520 525 Thr Val Met Val Gln Gly Val Ile Trp Asp Ile Asn Ser Phe Asp Gln 530 535 540 Trp Gly Val Glu Leu Gly Lys Gln Gln Ala Asn Asp Leu Ala Pro Ala 545 550 555 560 Val Ser Gly Glu Glu Asp Val Asp Ser Gly Asp Ser Ser Thr Asp Ser 565 570 575 Leu Ile Lys Trp Tyr Arg Ala Asn Arg 580 585 

What is claimed is:
 1. An isolated polynucleotide sequence, which encodes a polypeptide having an amino acid sequence that is at least 70% identical to a polypeptide having an at least one amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, and SEQ ID NO. 6, wherein the polypeptide has phosphoglucose isomerase activity.
 2. The isolated polynucleotide sequence of claim 1, wherein the encoded polypeptide has an amino acid sequence that is at least 80% identical to a polypeptide having an at least one amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, and SEQ ID NO.
 6. 3. The isolated polynucleotide sequence of claim 1, wherein the encoded polypeptide has an amino acid sequence that is at least 90% identical to a polypeptide having an at least one amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, and SEQ ID NO.
 6. 4. An isolated polypeptide having an amino acid sequence that is at least 70% identical to a polypeptide having an at least one amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, and SEQ ID NO. 6, wherein the polypeptide has phosphoglucose isomerase activity.
 5. The isolated polynucleotide sequence of claim 4, wherein the polypeptide has an amino acid sequence that is at least 80% identical to a polypeptide having an at least one amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, and SEQ ID NO.
 6. 6. The isolated polynucleotide sequence of claim 4, wherein the polypeptide has an amino acid sequence that is at least 90% identical to a polypeptide having an at least one amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, and SEQ ID NO.
 6. 7. The isolated polypeptide according to claim 4, wherein the polypeptide has a length of 585 amino acids.
 8. The isolated polypeptide according to claim 4, wherein the polypeptide has an N-terminus that is shortened by from 1 or 2, from 3 to 17, from 18 to 21, from 22 to 36 or from 37 to 46 amino acid residues.
 9. The isolated polypeptide according to claim 4, wherein the polypeptide has an N-terminus that is shortened by from 1 or 2, from 3 to 17, from 18 to 21, from 22 to 36 or from 37 to 46 amino acid residues and the C-terminus having an amino acid sequence selected from the group consisting of amino acids 47 to 585 of SEQ ID NO:2, amino acids 47 to 585 of SEQ ID NO:4, and amino acids 47 to 585 of SEQ ID NO:6.
 10. The isolated polypeptide according to claim 4, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:2, amino acids 2-585 of SEQ ID NO:2, SEQ ID NO:4, amino acids 2-585 of SEQ ID NO:4, SEQ ID NO:6, and amino acids 2-585 of SEQ ID NO:6.
 11. A host cell, comprising the isololated polypeptide of claim
 4. 12. The host cell of claim 11, which is a Corynebacterium glutamicum.
 13. The host cell of claim 11, wherein the phosphoglucose isomerase activity is enhanced or attenuated.
 14. A vector comprising the isolated polynucleotide of claim
 1. 15. A host cell comprising the isolated polynucleotide sequence of claim
 1. 16. The host cell of claim 14, which is a Corynebacterium glutamicum.
 17. The host cell of claim 14, wherein the phosphoglucose isomerase activity are present in enhanced or attenuated form
 18. A process for the production of amino acids, comprising a) enhancing the production of the polypeptides according to claim 4 in a host cell, b) fermenting the host cell, c) concentrating the amino acids in a medium, or in the fermentation liquor, or in the host cell, and d) isolating the amino acids, constituents of the fermentation liquor and/or biomass in totality or in part remaining in the product.
 19. The process according to claim 18, wherin the host cell is Corynebacterium glutamicum.
 20. The process according to claim 18, wherein the amino acids are one or more amino acids selected from the group consisting of L-lysine, L-valine, L-threonine, L-methionine, and mixtures thereof.
 21. The process according to claim 18, characterised in that the amino acids are L-lysine.
 22. A process for the production of amino acids, comprising a) enhancing the production of the polypeptides according to claim 4 in a host cell, b) fermenting the host cell, c) concentrating the amino acids in a medium, or in the fermentation liquor, or in the host cell, and d) isolating the amino acids, constituents of the fermentation liquor and/or biomass in totality or in part remaining in the product.
 23. The process according to claim 22, wherein the host cell is Corynebacterium glutamicum.
 24. The process according to claim 22, wherein the amino acids are one or more amino acids selected from the group consisting of L-tryptophan, L-phenylalanine, L-tyrosine, and mixtures thereof. 